# Patent

## Title
Hybrid enzymes

## Abstract

The present invention relates to hybrid polypeptides having a first amino acid sequence having endo-amylase activity and a second amino acid sequence having carbohydrate binding activity. The present invention also relates to the use of the hybrid polypeptides in starch processing and baking.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/315,801 filed on Dec. 22, 2005, now abandoned, which claims, under 35 U.S.C. 119, priority of Danish application nos. PA 2004 01976 and PA 2005 01261 filed on Dec. 22, 2004 and Sep 9, 2005, respectively, and the benefit of U.S. provisional application Nos. 60/639,181 and 60/717,274 filed on Dec. 22, 2004 and Sep. 14, 2005, respectively, the contents of which are fully incorporated herein by reference.


SEQUENCE LISTING
This application contains a sequence listing. A computer readable form containing the sequence listing accompanies this application, and the computer readable form of the sequence listing is hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates, inter alia, to hybrid enzymes comprising a carbohydrate binding module and having endo-amylase activity. The enzymes may be applied in processes comprising starch modification and/or degradation, or in dough making processes.
BACKGROUND OF THE INVENTION
Bacterial endo-amylases are used in a large number of processes, e.g., for liquefaction of starch in processes where starch is modified, and/or degraded to smaller polymers or monomers of glucose. The degradation products may used in the industry, e.g., as maltose and/or fructose syrups or further processed in a fermentation step to a fermentation product, e.g., ethanol. The bacterial endo-amylases are used in baking to give additional softness and a better moistness of the bread crumb. However, the endo-amylases are easy to overdose which may results in gumminess and an undesirable loss in elasticity in the baked product.
There is a need for endo-amylases with improved properties for use in various processes, e.g., within starch processing and baking.
SUMMARY OF THE INVENTION
The present inventors have now surprisingly discovered that by addition of a carbohydrate binding module (CBM) to an endo-amylase the catalytic activity of the endo-amylase can be modified thereby resulting in an increased baking performance compared to the wild type enzyme. There is no significant change in the taste or smell of the baked product. Without being bound by theory it is suggested that the effect is due to an increased activity towards raw starch in the dough conferred by the CBM, and/or a reduced activity towards the heated starch in the baking bread conferred by the CBM. The endo-amylase with a CBM can be used as a baking enzyme with less risk of overdosing compared to the enzyme without a CBM. Such hybrids consisting of a polypeptide having endo-amylase activity and a carbohydrate binding module, primarily having affinity for starch like, e.g., the CBM20, have the advantage over existing endo-amylases that by selecting a catalytic domain with desire properties, e.g., the pH profile, the temperature profile, the oxidation resistance, the calcium stability, the substrate affinity or the product profile can be combined with a carbohydrate binding module with stronger or weaker binding affinities, e.g., specific affinities for amylose, specific affinities for amylopectin or affinities for specific structure in the carbohydrate. The hybrid may be used as a baking additive, e.g., as an anti-staling enzyme.
The present inventors have further surprisingly discovered that by adding a carbohydrate-binding module (CBM) to an endo-amylase the activity and specificity can be altered thereby increasing the efficacy of various starch degrading processes, e.g., comprising degradation of raw, e.g., ungelatinized starch as well as gelatinized starch. Due to the superior hydrolysis activity of these endo-amylases having a CBM the overall starch conversion process can be performed without having to gelatinize the starch, i.e., the endo-amylases having a CBM hydrolyses granular starch in a raw starch process as well as fully or partially gelatinized starch in a traditional starch process.
Accordingly the invention provides in a first aspect a polypeptide which polypeptide is a hybrid comprising; a first amino acid sequence having endo-amylase activity and a second amino acid sequence comprising a carbohydrate-binding module. Preferably said first amino acid sequence and/or said amino acid second sequence is derived from a bacterium. The second amino acid sequence has preferably at least 60% identity to the amino acid sequence shown as amino acid residues 485 to 586 in SEQ ID NO:2 and/or the first amino acid sequence has at least 60% identity to the amino acid sequence shown in SEQ ID NO:35.
In a second aspect the invention provides a process for preparing a dough or an edible product made from a dough, which process comprises adding the polypeptide of the first aspect to a dough.
In a third and a fourth aspect the invention provides a composition comprising the polypeptide of the first aspect, and a dough- or bread-improving additive in the form of a granulate or agglomerated powder comprising the polypeptide of the first aspect.
In a fifth aspect the invention provides a process for designing a polypeptide suitable for baking, said process comprising; providing a first amino acid sequence having endo-amylase activity, and a second amino acid sequence comprising a carbohydrate-binding module; wherein said first amino acid sequence is derived from a bacterium; providing a second amino acid sequence comprising a carbohydrate-binding module; and constructing a polypeptide comprising said first amino acid sequence with said second amino acid sequence.
In a sixth aspect the invention provides a process for preparing composition, e.g., a bread improving additive, is produced in a process comprising the steps of; a) providing a first amino acid sequence having endo-amylase activity; b) providing a second amino acid sequence comprising a carbohydrate-binding module; c) and constructing a polypeptide comprising said first amino acid sequence and second amino acid sequence; d) providing a DNA sequence encoding said polypeptide; e) expressing said DNA sequence in a suitable host cell and recovering said polypeptide; f) adding said polypeptide to flour or to a granulate or agglomerated powder.
In a seventh aspect the invention provides a process for preparing a dough or an edible product made from a dough, which process comprises; providing a first amino acid sequence having endo-amylase activity; providing a second amino acid sequence comprising a carbohydrate-binding module; and constructing a polypeptide comprising said first amino acid sequence and second amino acid sequence; providing a DNA sequence encoding said polypeptide; expressing said DNA sequence in a suitable host cell and recovering said polypeptide; and adding said polypeptide to a dough.
In a eighth aspect the invention provides a process for saccharifying starch, wherein a starch is treated with the polypeptide according to the first aspect.
In a ninth aspect the invention provides a process comprising; contacting a starch with a polypeptide comprising a first amino acid sequence having endo-amylase activity, and a second amino acid sequence comprising a carbohydrate-binding module; wherein said first amino acid sequence and/or said second amino acid sequence is derived from a bacterium; incubating said starch with said polypeptide for a time and at a temperature sufficient to achieve conversion of at least 90% w/w of said starch substrate into fermentable sugars; fermenting to produce a fermentation product, and optionally recovering the fermentation product, wherein said polypeptide may be a polypeptide according to the first aspect.
In a tenth aspect the invention provides a process comprising; a) contacting a starch substrate with a yeast cell transformed to express a polypeptide comprising a first amino acid sequence having endo-amylase activity, and a second amino acid sequence comprising a carbohydrate-binding module; b) holding said starch substrate with said yeast for a time and at a temperature sufficient to achieve conversion of at least 90% w/w of said starch substrate into fermentable sugars; c) fermenting to produce ethanol; optionally recovering ethanol; wherein steps a, b, and c are performed separately or simultaneously and wherein said polypeptide may be a polypeptide according to the first aspect
In an eleventh aspect the invention provides a process of producing ethanol from starch-containing material by fermentation, said process comprises: a) liquefying said starch-containing material with a polypeptide comprising a first amino acid sequence having endo-amylase activity, and a second amino acid sequence comprising a carbohydrate-binding module; wherein said first amino acid sequence and/or second amino acid sequence is derived from a bacterium; b) saccharifying the liquefied mash obtained; c) fermenting the material obtained in step (b) in the presence of a fermenting organism.
In still further aspects the invention provides a DNA sequence encoding a polypeptide according to the first aspect, a DNA construct comprising said DNA sequence, a recombinant expression vector which carries said DNA construct, a host cell which is transformed with said DNA construct or said vector, said host cell being a bacterium or a fungal cell, a plant cell, or a yeast cell.


DETAILED DESCRIPTION OF THE INVENTION
Hybrid Enzymes
The polypeptide of the invention may be a hybrid enzyme comprises a first amino acid sequence having endo-amylase activity, and a second amino acid sequence comprising a carbohydrate-binding module (CBM). The hybrid may be produced by fusion of a first DNA sequences encoding a first amino acid sequences and a second DNA sequences encoding a second amino acid sequences, or the hybrid may be produced as a completely synthetic gene based on knowledge of the amino acid sequences of suitable CBMs, linkers and catalytic domains.
The terms “hybrid enzyme” (also referred to as “fusion protein”, “hybrid”, hybrid polypeptide” or “hybrid protein) is used herein to characterize the polypeptides of the invention comprising a first amino acid sequence comprising at least one catalytic module having endo-amylase activity and a second amino acid sequence comprising at least one carbohydrate-binding module wherein the first and the second are derived from different sources. The term “source” being understood as e.g., but not limited, to a parent enzyme, or a variant thereof, e.g., an amylase or glucoamylase, or other catalytic activity comprising a suitable catalytic module and/or a suitable CBM and/or a suitable linker. However the CBM may also be derived from a polypeptide having no catalytic activity. The first and the second amino acid sequence may be derived from the same bacterial strain, from strains within the same species, from closely related species or less related organisms. Preferably the first and the second amino acid sequence of the hybrids derived from different sources, e.g., from different enzymes from the same strain and/or species, or e.g., from strains within different species.
Enzyme classification numbers (EC numbers) referred to in the present specification are in accordance with the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (www.chem.qmw.ac.uk/iubmb/enzyme/).
Hybrid enzymes as referred to herein include species comprising an amino acid sequence of an endo-amylase, i.e., an alpha-amylase (EC 3.2.1.1) which is linked (i.e., covalently bound) to an amino acid sequence comprising a carbohydrate-binding module (CBM). The hybrid enzyme is thus an enzyme capable of catalyzing hydrolysis of starch in an endo-fashion.
CBM-containing hybrid enzymes, as well as detailed descriptions of the preparation and purification thereof, are known in the art [see, e.g., WO 90/00609, WO 94/24158 and WO 95/16782, as well as Greenwood et al., 1994, Biotechnology and Bioengineering 44: 1295-1305]. They may, e.g., be prepared by transforming into a host cell a DNA construct comprising at least a fragment of DNA encoding the carbohydrate-binding module ligated, with or without a linker, to a DNA sequence encoding the enzyme of interest, and growing the transformed host cell to express the fused gene. The linker may be a bond (i.e., comprising 0 residues), or a short linking group comprising from about 2 to about 100 carbon atoms, in particular of from 2 to 40 carbon atoms. However, the linker is preferably a sequence of 0 amino acid residues (e.g., just a bond) or it is from about 2 to about 100 amino acid residues, more preferably of from 2 to 40 amino acid residues, such as from 2 to 15 amino acid residues. Preferably the linker is not sensitive to or at least has low sensitivity towards hydrolysis by a protease, which e.g., may be present during production of the hybrid and/or during the industrial application of the hybrid. The CBM in a hybrid enzyme of the type in question may be positioned C-terminally, N-terminally or internally in the hybrid enzyme. In an embodiment a polypeptide may comprise more than one CBM, e.g., two CBMs; one positioned C-terminally, the other N-terminally or the two CBMs in tandem positioned C-terminally, N-terminally or internally. However, polypeptides with more than two CBMs are equally contemplated.
Polypeptide Identity
The term polypeptide “identity” is understood as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman and Wunsch, 1970, Journal of Molecular Biology 48: 443-453. The following settings for amino acid sequence comparison are used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. The relevant part of the amino acid sequence for the identity determination is the mature polypeptide, i.e., without the signal peptide.
Carbohydrate-Binding Modules
A carbohydrate-binding module (CBM), or as often referred to, a carbohydrate-binding domain (CBD), is a polypeptide amino acid sequence which binds preferentially to a poly- or oligosaccharide (carbohydrate), frequently—but not necessarily exclusively—to a water-insoluble (including crystalline) form thereof.
CBMs derived from starch degrading enzymes are often referred to as starch-binding modules or SBMs (CBMs which may occur in certain amylolytic enzymes, such as certain glucoamylases, or in enzymes such as cyclodextrin glucanotransferases, or in endo-amylases). SBMs are often referred to as SBDs (Starch Binding Domains). Prefered for the invention are CBMs which are Starch Binding Modules.
CBMs are found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes (hydrolases) which typically comprise a catalytic module containing the active site for substrate hydrolysis and a carbohydrate-binding module (CBM) for binding to the carbohydrate substrate in question. Such enzymes can comprise more than one catalytic module and one, two or three CBMs, and optionally further comprise one or more polypeptide amino acid sequence regions linking the CBM(s) with the catalytic module(s), a region of the latter type usually being denoted a “linker”. CBMs have also been found in algae, e.g., in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein.
In proteins/polypeptides in which CBMs occur (e.g., enzymes, typically hydrolytic enzymes), a CBM may be located at the N or C terminus or at an internal position.
That part of a polypeptide or protein (e.g., hydrolytic enzyme) which constitutes a CBM per se typically consists of more than about 30 and less than about 250 amino acid residues.
The “Carbohydrate-Binding Module of Family 20” or a CBM-20 module is in the context of this invention defined as a sequence of approximately 100 amino acids having at least 45% identity to the Carbohydrate-Binding Module (CBM) of the polypeptide disclosed in FIG. 1 by Joergensen et al., 1997, Biotechnol. Lett. 19:1027-1031. The CBM comprises the last 102 amino acids of the polypeptide, i.e., the subsequence from amino acid 582 to amino acid 683. The numbering of Glycoside Hydrolase Families applied in this disclosure follows the concept of Coutinho, P. M. & Henrissat, B. (1999) CAZy—Carbohydrate-Active Enzymes server at URL: afmb.cnrs-mrs.fr/ ̃cazy/CAZY/index.html or alternatively Coutinho, P. M. & Henrissat, B. 1999; The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In “Genetics, Biochemistry and Ecology of Cellulose Degradation”, K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo, pp. 15-23, and Bourne, Y. & Henrissat, B. 2001; Glycoside hydrolases and glycosyltransferases: families and functional modules, Current Opinion in Structural Biology 11:593-600.
Examples of enzymes which comprise a CBM suitable for use in the context of the invention are endo-amylases (i.e., alpha-amylases in EC 3.2.1.1), maltogenic alpha-amylases (EC 3.2.1.133), glucoamylases (EC 3.2.1.3) or from CGTases (EC 2.4.1.19).
Preferred for the invention is CBMs of Carbohydrate-Binding Module Family 20. CBMs of Carbohydrate-Binding Module Family 20 suitable for the invention may be derived from beta-amylases of Bacillus cereus (SWISSPROT P36924), or from CGTases of Bacillus circulans (SWISSPROT P43379). Also preferred for the invention is any CBM having at least 60%, at least 70%, at least 80% or even at least 90% identity to any of the afore mentioned CBM amino acid sequences. Further suitable CBMs of Carbohydrate-Binding Module Family 20 may be found at URL: afmb.cnrs-mrs.fr/ ̃cazy/CAZY/index.html).
Once a nucleotide sequence encoding the substrate-binding (carbohydrate-binding) region has been identified, either as cDNA or chromosomal DNA, it may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding the enzyme of interest. The DNA fragment encoding the carbohydrate-binding amino acid sequence and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA may then be manipulated in a variety of ways to achieve expression.
CBMs deriving from bacteria will generally be suitable for use in the context of the invention, however, preferred are CBMs of bacillus origin, such as a CBM20 from Bacillus flavothermus (Syn. Anoxybacillus contaminans), preferably from amylase AMY1048 (SEQ ID NO:2 herein), AMY1039, or AMY1079 (disclosed as respectively SEQ ID NOS: 1, 2 and 3 in PCT/US2004/023031 [NZ10474]), the Bacillus amylases disclosed in WO 2002/068589 from Diversa, Bacillus sp. TS23 (Korea) (Lin, L.-L.; Submitted (1 Mar. 1995) to the EMBL/GenBank/DDBJ databases. Long-Liu Lin, Food Industry Research Institute, Culture
Collection and Research Center, 331 Food Road, Hsinchu, Taiwan 300, Republic of China).
In a particular embodiment the CBM sequence has the amino acid sequence shown as amino acid residues 485 to 586 in SEQ ID NO:2 or the CBM sequence has an amino acid sequence having at least 60%, at least 70%, at least 80% or even at least 90% identity to the afore mentioned amino acid sequence.
In another preferred embodiment the CBM sequence has an amino acid sequence which differs from the amino acid sequence shown as amino acid residues 485 to 586 in SEQ ID NO:2 in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.
Endo-Amylase Sequence
Endo-amylases which are appropriate as the basis for CBM/amylase hybrids of the types employed in the context of the present invention include those of bacterial origin and having endo-amylase activity. The endo-activity of the amylase may be determined according to the assay in the “Materials and methods” section of the present application. Preferred are endo-amylase derived from Bacillus sp., particularly from B. licheniformis, B. amyloliquefaciens, B. stearothermophilus or B. flavothermus. The endo-amylase is preferably an endo-amylase having at least 60%, at least 70%, at least 80% or even at least 90% identity to the amylase from Bacillus licheniformis (BLA, SEQ ID NO:8 in WO 2002/010355) shown in SEQ ID NO:35 herein. This includes but are not limited to the amylase from B. licheniformis variant LE429 (WO 2002/010355) shown in SEQ ID NO:41 herein, the amylase from B. stearothermophilus (BSG, SEQ ID NO:6 in WO 2002/010355) shown in SEQ ID NO:36 herein, the amylase from B. amyloliquefaciens (BAN, SEQ ID NO:10 in WO 2002/010355) shown in SEQ ID NO:37 herein, the amylase from B. halodurance SP722 (SEQ ID NO:4 in WO 2002/010355) shown in SEQ ID NO:38 herein, SP690 (WO 95/26397) shown in SEQ ID NO:39 herein, the amylase from AA560 (SEQ ID NO:12 in WO 2002/010355) shown in SEQ ID NO:40 herein, the amylase from alkaline Bacillus strains like e.g., SP707 (Tsukamoto et al., 1988, Biochemical and Biophysical Research Communications 151: 25-31), the amylase KSM-AP1378 (WO 97/00324, KAO), the amylases KSM-K36 and KSM-K38 (EP 1,022,334-A/KAO), the amylase SP7-7 (WO 02/10356, Henkel), and the amylase AAI-6 (WO 00/60058), AMRK385 (PCT/DK01/00133)—fragments, variants or truncated forms of above. The endo-amylase sequence may also be derived from Pseudomonas saccharophilia, such as from the amylase disclosed as SEQ ID NO:1 in WO 2004/111217. Preferably endo-amylase sequence comprises the amino acid residues 1 to 417 shown in SEQ ID NO:42 herein.
Preferably the endo-amylase is a wild type enzyme or the endo-amylase is a variant endo-amylases comprising amino acid modifications leading to increased activity and/or increased protein stability at low pH, and/or at high pH, increased stability towards calcium depletion, and/or increased stability at elevated temperature. Chemically or genetically modified mutants of such endo-amylases are included in this connection.
The B. licheniformis endo-amylase BLA shown in SEQ ID NO:35 is a wild type amylase made up of a catalytic fragment of 483 amino acid. The catalytic domain can be divided into the central core-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain. In Seq. ID 8/NN10062 the catalytic core domain consist of the first 396 amino acids and the C domain is defined as the amino acids from 397 to 483.
The variant of the B. licheniformis endo-amylase, LE429 shown in SEQ ID NO:41 consist of a catalytic fragment of 481 amino acid. The catalytic domain can be divided into the central core-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain. In SEQ ID NO:41 the catalytic core domain consist of the first 394 amino acids and the C domain is defined as the amino acids from 395 to 481.
The B. amyloliquefaciens endo-amylase, BAN shown in SEQ ID NO:37 is a wild type amylase made up of a catalytic fragment of 483 amino acid. The catalytic domain can be divided into the central core-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain. In SEQ ID NO:37 the catalytic core domain consists of the first 396 amino acids and the C domain is defined as the amino acids from 397 to 483.
The B. stearothermophilus endo-amylase, BSG shown in SEQ ID NO:36 is a wild type amylase made up of a catalytic fragment of 483 amino acid and in addition a c-terminal extension. The catalytic domain can further be divided into the central core-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain. In SEQ ID NO:36 the catalytic core domain consist of the first 396 aa, the C domain is defined as the amino acids from 397 to 483 and the c-terminal extension is defines as amino acids 484 to 515.
The B. halodurance endo-amylase SP722 shown in SEQ ID NO:38 is a wild type amylase made up of a catalytic fragment of 485 amino acid. The core domain can further be divided into the central AB-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain. In SEQ ID NO:38 the catalytic core domain consist of the first 398 amino acids and the C domain is defined as the amino acids from 399 to 485.
The alkaline Bacillus endo-amylase, AA560 shown in SEQ ID NO:40 herein is a wild type amylase made up of a catalytic fragment of 485 amino acid. The core domain can further be divided into the central AB-domain harboring the catalytic center and a C domain C-terminal to the catalytic domain. The catalytic core domain consists of the first 398 amino acids and the C domain is defined as the amino acids from 399 to 485. The catalytic core domain is encoded by nucleotide 1-1194 and the C domain is encoded by the nucleotides 1189-1455.
In a particular embodiment of the first aspect the endo-amylase sequence has the amino acid sequence shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 or the endo-amylase sequence has an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or even at least 99% identity to any of the afore mentioned amino acid sequences.
In yet another preferred embodiment of the first aspect the endo-amylase sequence has an amino acid sequence which differs from any of the amino acid sequence amino acid sequences shown in SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.
In a preferred embodiment of the first aspect the endo-amylase sequence has an amino acid sequence as shown in SEQ ID NO:40 (AA560), and comprising one or more of the following alterations R118K, D183*, G184*, N195F, R320K and R458K.
In another particularly preferred embodiment of the first aspect the endo-amylase sequence has an amino acid sequence as shown in SEQ ID NO:40, and comprising one or more, e.g., such as all, of the following alterations R118K, D183*, G184*, N195F, R320K, R458K, N33S, D36N, K37L, E391I, Q394R, K395D, T452Y and N484P.
In another particularly preferred embodiment of the first aspect the endo-amylase sequence has an amino acid sequence as shown in SEQ ID NO:40, and comprising one or more, e.g., such as all, of the following alterations R118K, D183*, G184*, N195F, R320K, R458K and N484P.
In yet another highly preferred embodiment of the first aspect the endo-amylase sequence has an amino acid sequence as shown in SEQ ID NO:37 and comprises one or more, e.g such as all of the following alterations: S31A, D32N, I33L, E178*, G179*, N190F, K389I, K392R, E393D, V508A
Preferred Hybrids
In a particular embodiment the hybrid of the invention has an amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or the hybrid of the invention has an amino acid sequence having at least 60%, at least 70%, at least 80% or even at least 90% identity to any of the aforementioned amino acid sequences.
In yet another preferred embodiment the hybrid of the invention has an amino acid sequence which differs from the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.
In a preferred embodiment the polypeptide of the invention comprises a) the catalytic domain shown in SEQ ID NO:40 or a homologous catalytic domain, and b) the CBM shown as residue 485 to 585 of SEQ ID NO:2, wherein one or more, or preferably all, of the following substitutions have been introduced: R118K, D183*, G184*, N195F, R320K, R458K, N33S, D36N, K37L, E391I, Q394R, K395D, T452Y and N484P, using the numbering of SEQ ID NO: 40.
In another preferred embodiment the polypeptide of the invention comprises the catalytic domain shown in SEQ ID NO:40 or a homologous catalytic domain, and b) the CBM shown as residue 485 to 585 of SEQ ID NO:2, wherein one or more, or preferably all, of the following substitutions have been introduced: R118K, D183*, G184*, N195F, R320K, R458K and N484P, using the numbering of SEQ ID NO: 40.
In yet another preferred embodiment the polypeptide of the invention comprises the catalytic domain shown in SEQ.ID: 37 and comprise one or more, e.g., such as all of the following alterations: S31A, D32N, I33L, E178*, G179*, N190F, K389I, K392R, E393D, V508A and a CBM having the amino acid sequence shown as amino acid residues 485 to 586 in SEQ ID NO:2.
Stabilization of Hybrids
A hybrid of the invention may be volatile to proteolytic attack if the CBM and catalytic domain proteins do not form sufficiently tight protein-protein interactions. However, the stability of the hybrid can be improved by introducing substitutions on the surface of either of the proteins to create a stable hybrid.
The present inventors have identified the following amino acid residues on the surface of bacterial endo-amylases, e.g., such polypeptides having at least 60% identity to the amylase from Bacillus licheniformis (SEQ ID NO:8), to be in close contact with the CBM when comprised in the hybrid of the invention, i.e., within less than 5.0 Å distance. These residues are suitable targets for mutations in order to make a stable hybrid: 12, 29, 30, 32, 33, 34, 35, 36, 37, 38, 368, 371, 372, 381, 383, 384, 386, 387, 388, 389, 390, 391, 392, 394, 395, 396, 422, 423, 448, 449, 450, 451, 452, 453, 454, 455, 456, 458, 459, 460, 461, 483, 484, 485 using the numbering of SEQ ID NO: 40. Preferably the catalytic domain of the hybrid of the invention comprises one or more substitutions in positions corresponding to these residues.
In a preferred embodiment the hybrid of the invention comprises a) the catalytic domain shown in SE ID NO:40 or a homologous catalytic domain, and b) the CBM shown as residue 485 to 585 of SEQ ID NO:2, wherein one or more, or preferably all, of the following substitutions have been introduced: N33S, K35S/A, D36A/N/S, K37L, E391I, Q394R, K395D, N484A/P using the numbering of SEQ ID NO: 40.
On the surface of the CBM protruding towards the catalytic domain of the hybrid the following residues are found in close contact with the catalytic domain, i.e., within 5.0 Å distance, and these residues are suitable targets for mutations in order to make a stable hybrid: 485, 486, 487, 488, 507, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 526, 538, 539, 540, 541, 553, 554, 555, 556, 557, 558, 559 using the numbering of SEQ ID NO: 2.
Expression Vectors
The present invention also relates to recombinant expression vectors which may comprise a DNA sequence encoding the hybrid enzyme, a promoter, a signal peptide sequence, and transcriptional and translational stop signals. The various DNA and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the DNA sequence encoding the polypeptide at such sites. Alternatively, the DNA sequence of the present invention may be expressed by inserting the DNA sequence or a DNA construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression, and possibly secretion.
The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the DNA sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, a cosmid or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. The vector system may be a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
Host Cells
The host cell of the invention, either comprising a DNA construct or an expression vector comprising the DNA sequence encoding the polypeptide of the first aspect, e.g., a hybrid enzyme, is advantageously used as a host cell in the recombinant production of the hybrid enzyme, wild type enzyme or a genetically modified wild type enzyme. The cell may be transformed with an expression vector. Alternatively, the cell may be transformed with the DNA construct of the invention encoding the hybrid enzyme or a genetically modified wild type enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. Integration of the DNA construct into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination.
The host cell may be any appropriate prokaryotic or eukaryotic cell, e.g., a bacterial cell, a filamentous fungus cell, a yeast cell, a plant cell or a mammalian cell.
Isolating and Cloning a DNA Sequence Encoding a Parent Endo-Amylase
The techniques used to isolate or clone a DNA sequence encoding the polypeptide of the first aspect, e.g., a hybrid enzyme, are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the DNA sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other DNA amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and DNA sequence-based amplification (NASBA) may be used.
The DNA sequence encoding a parent endo-amylase may be isolated from any cell or microorganism producing the endo-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the endo-amylase to be studied. Then, if the amino acid sequence of the endo-amylase is known, labeled oligonucleotide probes may be synthesized and used to identify endo-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known endo-amylase gene could be used as a probe to identify endo-amylase-encoding clones, using hybridization and washing conditions of very low to very high stringency.
Yet another method for identifying endo-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming endo-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for endo-amylase (i.e., maltose), thereby allowing clones expressing the endo-amylase to be identified.
Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described Beaucage and Caruthers, 1981, Tetrahedron Letters 22: 1859-1869, or the method described by Matthes et al., 1984, EMBO J. 3: 801-805. In the phosphoroamidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., 1988, Science 239: 487-491.
Isolated DNA Sequence
The present invention relates, inter alia, to an isolated DNA sequence comprising a DNA sequence encoding a polypeptide of the first aspect, e.g., a hybrid enzyme.
The term “isolated DNA sequence” as used herein refers to a DNA sequence, which is essentially free of other DNA sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis.
For example, an isolated DNA sequence can be obtained by standard cloning procedures used in genetic engineering to relocate the DNA sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired DNA fragment comprising the DNA sequence encoding the polypeptide of interest, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the DNA sequence will be replicated. An isolated DNA sequence may be manipulated in a variety of ways to provide for expression of the polypeptide of interest. Manipulation of the DNA sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying DNA sequences utilizing recombinant DNA methods are well known in the art.
DNA Construct
The present invention relates, inter alia, to a DNA construct comprising a DNA sequence encoding a polypeptide of the first aspect. “DNA construct” is defined herein as a DNA molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of DNA, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. The term DNA construct is synonymous with the term expression cassette when the DNA construct contains all the control sequences required for expression of a coding sequence of the present invention.
Site-Directed Mutagenesis
Once a parent endo-amylase-encoding DNA sequence suitable for use in a polypeptide of the first aspect has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. In a specific method, a single-stranded gap of DNA, the endo-amylase-encoding sequence, is created in a vector carrying the endo-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al., 1984, Biotechnology 2: 646-639. U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.
Another method for introducing mutations into endo-amylase-encoding DNA sequences is described in Nelson and Long, 1989, Analytical Biochemistry 180: 147-151. It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
Localized Random Mutagenesis
The random mutagenesis may be advantageously localized to a part of the parent endo-amylase in question. This may, e.g., be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.
The localized or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above.
Expression of the Enzymes in Plants
A DNA sequence encoding an enzyme of interest, such as a hybrid enzyme of the present invention, may be transformed and expressed in transgenic plants as described below.
The transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot. Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum and maize (corn).
Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana. 
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. In the present context, also specific plant cell compartments, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts e.g., embryos, endosperms, aleurone and seeds coats.
Also included within the scope of the invention are the progeny of such plants, plant parts and plant cells.
The transgenic plant or plant cell expressing the enzyme of interest may be constructed in accordance with methods known in the art. In short the plant or plant cell is constructed by incorporating one or more expression constructs encoding the enzyme of interest into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
Conveniently, the expression construct is a DNA construct which comprises a gene encoding the enzyme of interest in operable association with appropriate regulatory sequences required for expression of the gene in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, e.g., on the basis of when, where and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding the enzyme of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific cell compartment, tissue or plant part such as seeds or leaves. Regulatory sequences are, e.g., described by Tague et al., 1988, Plant Phys. 86: 506.
For constitutive expression the 35S-CaMV, the maize ubiquitin 1 and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294, Christensen, Sharrock and Quail, 1992 Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18: 675-689; Zhang, McElroy and Wu, 1991, Analysis of rice Act1 5′ region activity in transgenic rice plants. Plant Cell 3: 1155-1165). Organ-specific promoters may, e.g., be a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Annu. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39(8): 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba described by Conrad et al, 1998, Journal of Plant Physiology 152(6): 708-711, a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39(9): 935-941, the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102(3): 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26(1): 85-93, or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248(6): 668-674, or a wound inducible promoter such as the potato pin2 promoter (Xu et al, 1993, Plant Molecular Biology 22(4): 573-588. Likewise, the promoter may inducible by abiotic treatments such as temperature, drought or alterations in salinity or induced by exogenously applied substances that activate the promoter e.g., ethanol, oestrogens, plant hormones like ethylene, abscisic acid and gibberellic acid and heavy metals.
A promoter enhancer element may be used to achieve higher expression of the enzyme in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding the enzyme. For instance, Xu et al. op cit disclose the use of the first intron of the rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
The DNA construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science 244: 1293; Potrykus, 1990, Bio/Techn. 8: 535; Shimamoto et al., 1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens mediated gene transfer is the method of choice for generating transgenic dicots (for review Hooykas & Schilperoort, 1992, Plant Mol. Biol. 19: 15-38), and can also be used for transforming monocots, although other transformation methods often are used for these plants. Presently, the method of choice for generating transgenic monocots supplementing the Agrobacterium approach is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21(3): 415-428.
Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well-known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using e.g., co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.
Dough-Based Products
The hybrid enzyme of the present invention may be used for the preparation of a dough-based edible product such as, bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, more preferably baked products, such as, bread products.
The dough used to prepare the dough based product generally comprises flour, e.g., from grains, such as, wheat flour, corn flour, rye flour, oat flour, or sorghum flour. The dough is generally leavened by the addition of a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast) or a chemical leavening agent.
The edible dough based product may preferably be any kind of baked product prepared from dough, either of a soft or a crisp character, either of a white, light or dark type. Preferred edible dough based products include bread (in particular white, wheat, whole-meal, low-carb, brown, multi-grain, dark and rye bread), typically in the form of loaves, buns or rolls, and more preferably, pan bread, hamburger buns, French baguette-type bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie crusts, crisp bread, steamed bread, pizza crust and the like.
The edible dough-based product is made by heating the dough, e.g., by baking or steaming. Examples are steamed or baked bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls. The edible dough based product may also be prepared by frying (e.g., deep frying in hot fat or oil). An example of such an edible product is a doughnut.
The hybrid enzymes of the first aspect of the invention preferably have a high tolerance towards overdosing. The addition of the polypeptide of the invention, e.g., the polypeptide of the first aspect, in 2 times, 3 times, preferably 4 times, more preferably 5 times, most preferably 6 times the effective dosage of said polypeptide to a dough results in an ELR and/or an ELRN of less than 15%, less than 10%, less than 7%, less than 6%, less than 5%, less than 4% or even less than 3%.
In a further aspect the polypeptide of the invention has a residual activity of at least 20%, such as at least 25% or 30%, preferably at least 35%, more preferably at least 40% and most preferably at least 50%, at the test conditions given in the specification.
The polypeptide of the present invention may further have an improved exo-to-endo ratio de-fined as IEF1 or IEF2 in the specification. The IEF1 or IEF2 of the polypeptide may be larger than 1, such as 1.1 or 1.5, preferably 2 or 2.5 or 3, more preferably 3.5 or 4, most preferably 5 or 7 or 10.
In further embodiments the invention provides polypeptides with characteristics that are of particular interest for baking purposes, namely a residual activity of at least 25% at 70° C. at the test conditions given in the specification, an increased exo-to-endo ratio (IEF), where IEF is larger than 1, and finally a reduced cohesiveness of less than 5% (at the test conditions given in the specification) while change in hardness is at least 85 units (at the test conditions given in the specification) and/or change mobility of free water is at least 1100 units (at the test conditions given in the specification).
For baking purpose the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 5%, while dHardness, when measured at the test conditions given in the specification, is at least 85 units, such as 90 units or 100 units, preferably 150 units or 200 units, more preferably 250 units or 300 units, most preferably 400 units or 600 units. In a further embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 4%, while dHardness, when measured at the test conditions given in the specification, is at least 85 units, such as 90 units or 100 units, preferably 150 units or 200 units, more preferably 250 units or 300 units, most preferably 400 units or 600 units. In a still further embodiment the polypeptide of the invention may give a cohesive-ness reduction, when measured at the test conditions given in the specification, of at least 2%, while dHardness, when measured at the test conditions given in the specification, is at least 85 units, such as 90 units or 100 units, preferably 150 units or 200 units, more preferably 250 units or 300 units, most preferably 400 units or 600 units. In yet another embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 1%, while dHardness, when measured at the test conditions given in the specification, is at least 85 units, such as 90 units or 100 units, preferably 150 units or 200 units, more preferably 250 units or 300 units, most preferably 400 units or 600 units.
When the polypeptide of the invention is added together with 300 MANU Novamyl®/kg flour it may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 5%, while dHardness, when measured at the test conditions given in the specification, is at least 15 units, such as 20 units or 30 units, preferably 40 units or 50 units, more preferably 60 units or 70 units, most preferably 85 units or 100 units. In a further embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 4%, while dHardness, when measured at the test conditions given in the specification, is at least 15 units, such as 20 units or 30 units, preferably 40 units or 50 units, more preferably 60 units or 70 units, most preferably 85 units or 100 units. In a still further embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 2%, while dHardness, when measured at the test conditions given in the specification, is at least 15 units, such as 20 units or 30 units, preferably 40 units or 50 units, more preferably 60 units or 70 units, most preferably 85 units or 100 units. In yet another embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 1%, while dHardness, when measured at the test conditions given in the specification, is at least 15 units, such as 20 units or 30 units, preferably 40 units or 50 units, more preferably 60 units or 70 units, most preferably 85 units or 100 units.
For baking purpose the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 5%, while dMobility, when measured at the test conditions given in the specification, is at least 300 units, such as 400 units or 500 units, preferably 600 units or 700 units, more preferably 800 units or 900 units, most preferably 1000 units or 1200 units. In a further embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 4%, while dMobility, when measured at the test conditions given in the specification, is at least 300 units, such as 400 units or 500 units, preferably 600 units or 700 units, more preferably 800 units or 900 units, most preferably 1000 units or 1200 units. In a still further embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 2%, while dMobility, when measured at the test conditions given in the specification, is at least 300 units, such as 400 units or 500 units, preferably 600 units or 700 units, more preferably 800 units or 900 units, most preferably 1000 units or 1200 units. In yet another embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 1%, while dMobility, when measured at the test conditions given in the specification, is at least 300 units, such as 400 units or 500 units, preferably 600 units or 700 units, more preferably 800 units or 900 units, most preferably 1000 units or 1200 units.
When the polypeptide of the invention is added together with 300 MANU Novamyl®/kg flour it may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 5%, while dMobility, when measured at the test conditions given in the specification, is at least 1000 units, such as 1100 units or 1200 units, preferably 1400 units or 1500 units, more preferably 1800 units or 2000 units, most preferably 2200 units or 2500 units. In a further embodiment the polypeptide of the invention may give a cohesive-ness reduction, when measured at the test conditions given in the specification, of at least 4%, while dMobility, when measured at the test conditions given in the specification, is at least 1000 units, such as 1100 units or 1200 units, preferably 1400 units or 1500 units, more preferably 1800 units or 2000 units, most preferably 2200 units or 2500 units. In a still further embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 2%, while dMobility, when measured at the test conditions given in the specification, is at least 1000 units, such as 1100 units or 1200 units, preferably 1400 units or 1500 units, more preferably 1800 units or 2000 units, most preferably 2200 units or 2500 units. In yet another embodiment the polypeptide of the invention may give a cohesiveness reduction, when measured at the test conditions given in the specification, of at least 1%, while dMobility, when measured at the test conditions given in the specification, is at least 1000 units, such as 1100 units or 1200 units, preferably 1400 units or 1500 units, more preferably 1800 units or 2000 units, most preferably 2200 units or 2500 units.
The above values for cohesiveness reduction, dHardness and dMobility are particularly relevant for bread, in particular for bread prepared by the sponge and dough method. Similar correlation between cohesiveness reduction and dHardness and dMobility is disclosed in Example 7.
The hybrid enzyme of the present invention may optionally be used together with one or more additional enzymes and/or anti-staling agents.
Anti-staling agents include but are not limited to emulsifiers, hydrocolloids and enzymatic anti-staling agents. As used herein, an anti-staling agent refers to a chemical, biological or enzymatic agent which can retard staling of the dough-based products, that is, which can reduce the rate deterioration of the softness of the dough based product during storage. The softness of dough based products (and the anti-staling effect of the anti-staling agent) can be evaluated empirically by the skilled test baker or measured using a texture analyzer (e.g., TAXT2), as is known in the art.
Examples of chemical anti-staling agents include polar lipids, e.g., fatty acids and their monoglyceride esters, such as, described in U.S. Pat. No. 4,160,848.
In a preferred embodiment, the anti-staling agent is an anti-staling enzyme, which is preferably added to the dough prior to cooking (e.g., baking). Examples of anti-staling enzymes include, without limitation, endo-amylases, such as the hybrids of the invention, exo-endo-amylases, such as, e.g., the exo-amylase described in U.S. Pat. No. 6,667,065 and US 2004/0043109, pullulanases, glycosyltransferases, amyloglycosidases, branching enzymes (1,4-alpha-glyucan branching enzyme), 4-alpha-glucanotransferases (dextrin transferase), beta-amylases, maltogenic alpha-amylases, lipases, phospholipases, galactolipases, acyltransferases, pectate lyases, xylanases, xyloglucan endotransglycosylases, proteases, e.g., as described in WO 2003/084331, peptidases and combinations thereof. The amylase may be from a fungus, bacterium or plant. It may be an endo-amylase, e.g., from Bacillus, particularly B. licheniformis or B. amyloliquefaciens, a beta-amylase, e.g., from plant (e.g., soy bean) or from microbial sources (e.g., Bacillus), such as the non-maltogenic Bacillus clausii alpha-amylase disclosed in WO 99/50399A2, the Pseudomonas saccharophilia amylase in SEQ ID NO:1 of WO 2004/111217, or a glucoamylase, or a fungal endo-amylase, e.g., from A. niger or A. oryzae. 
More preferably, the additional enzyme is an anti-staling enzyme and preferably the anti-staling enzyme is a maltogenic amylase (EC 3.2.1.133). The maltogenic amylases is added into the dough in an amount effective to retard the staling of the product, such as, at least 500 MANU/kg flour, more preferably in an amount of at least 500 to 1500 MANU/kg flour. A maltogenic amylase may be obtained from any suitable source, such as derived from a bacteria, such as Bacillus, preferably B. stearothermophilus, e.g., from strain NCIB 11837 or a variant thereof made by amino acid modification (EP 494233 B1, U.S. Pat. No. 6,162,628). The maltogenic amylase may preferably be added at a dosage of 20 to 2000 MANU/kg flour, preferably 500 to 1000 MANU/kg flour, more preferably, at least 750 MANU/kg flour, at least 1000 MAN U/kg flour. A preferred maltogenic amylase is Novamyl® (available form Novozymes A/S).
In another preferred embodiment, the anti-staling enzyme is a xylanase. The xylanase may be obtained from any suitable source, e.g., from Bacillus, e.g., Bacillus subtilis, as described in WO 2003/010923, WO 2001/066711 or WO 2000/039289, and Aspergillus, in particular of A. aculeatus, A. niger, A. awamori, or A. tubigensis or Trichoderma and Thermomyces as described in WO 96/32472, e.g., T. reesei, or from a strain of Humicola, e.g., H. insolens. Optionally, an additional enzyme may be used together with the above anti-staling enzymes, such as, a lipolytic enzyme, particularly phospholipase, galactoilipase and/or triacyl glycerol lipase activity, e.g., as described in WO 99/53769, WO 00/32758, WO 02/00852 or WO 2002/066622, or e.g., a transglutaminase, a cellulytic enzyme, e.g., a cellulase, an acyltransferase, a protein disulfide isomerase, a pectinase, a pectate lyase, an oxidoreductase. The enzyme may be of any origin, including mammalian, plant, and preferably microbial (bacterial, yeast or fungal) origin and may be obtained by techniques conventionally used in the art.
The additional enzyme may also be a lipolytic enzyme, particularly phospholipase, galactoilipase and/or triacyl glycerol lipase activity, e.g., as described in WO 99/53769, WO 00/32758, WO 02/00852 or WO 2002/066622.
Further, the additional enzyme may be a second amylase, a cyclodextrin glucanotransferase, a protease or peptidase, in particular an exopeptidase, a trans-glutaminase, a lipase, a phospholipase, a cellulase, a hemicellulase, a glycosyltransferase, a branching enzyme (1,4-alpha-glucan branching enzyme) or an oxidoreductase. The additional enzyme may be of mammalian, plant or microbial (bacterial, yeast or fungal) origin.
The second amylase may be from a fungus, bacterium or plant. It may be a maltogenic amylase (EC 3.2.1.133), e.g., from B. stearothermophilus, an endo-amylase, e.g., from Bacillus, particularly B. licheniformis or B. amyloliquefaciens, a beta-amylase, e.g., from plant (e.g., soy bean) or from microbial sources (e.g., Bacillus), a glucoamylase, e.g., from A. niger, or a fungal endo-amylase, e.g., from A. oryzae or from Pseudomonas saccharophilia such as the non-maltogenic alpha-amylase disclosed in WO 99/50399A2.
The hemicellulase may be a pentosanase, e.g., a xylanase which may be of microbial origin, e.g., derived from a bacterium or fungus, such as a strain of Aspergillus, in particular of A. aculeatus, A. niger, A. awamori, or A. tubigensis, from a strain of Trichoderma, e.g., T. reesei, or from a strain of Humicola, e.g., H. insolens. 
The protease may be from Bacillus, e.g., B. amyloliquefaciens. 
The oxidoreductase may be a glucose oxidase, a carbohydrate oxidase, a hexose oxidase, a lipoxidase, a peroxidase, or a laccase.
Dough and/or Bread-Improving Additive
The hybrid enzyme of the present invention may be provided as a dough and/or bread improving additive in the form of a granulate or agglomerated powder. The dough and/or bread improving additive may preferably have a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 μm.
In a preferred embodiment a composition, e.g., a bread improving additive, is produced in a process comprising the steps of; a) providing a first amino acid sequence having endo-amylase activity; b) providing a second amino acid sequence comprising a carbohydrate-binding module; c) and constructing a polypeptide comprising said first amino acid sequence and second amino acid sequence; d) providing a DNA sequence encoding said polypeptide; e) expressing said DNA sequence in a suitable host cell and recovering said polypeptide; f) adding said polypeptide to flour or to a granulate or agglomerated powder.
Granulates and agglomerated powders may be prepared by conventional methods, e.g., by spraying the amylase, i.e., the hybrid enzyme, onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g., a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.
Starch Processing
The polypeptide of this invention, i.e., an endo-amylase having a CBM, possesses valuable properties allowing for a variety of industrial applications. In particular, enzymes of the first aspect are applicable as a component in washing, dishwashing and hard-surface cleaning detergent compositions. Numerous variants are particularly useful in the production of sweeteners and ethanol from starch, and/or for textile desizing. One example of producing ethanol, wherein an endo-amylase of the invention may be used is disclosed in U.S. Pat. No. 5,231,017 which is hereby incorporated by reference.
Further, a process wherein an endo-amylase of the invention may be used is disclosed in DK patent application no. PA 2003 01568 (hereby incorporated by reference). Said process comprises hydrolysing starch into a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of said granular starch. Another suitable process is disclosed in WO 2004/081193 (hereby incorporated by reference).
Conditions for conventional starch-conversion processes, including starch liquefaction and/or saccharification processes are described in, e.g., U.S. Pat. No. 3,912,590 and in EP patent publications Nos. 252,730 and 63,909.
A preferred use is in a fermentation process wherein a starch substrate is liquefied and/or saccharified in the presence of the endo-amylase having a CBM to produce glucose and/or maltose, e.g., for use as sweeteners or suitable for conversion into a fermentation product by a fermenting organism, preferably a yeast. Such fermentation processes include a process for producing ethanol for fuel or drinking ethanol (portable alcohol), a process for producing a beverage, a process for producing organic compounds, such as citric acid, itaconic acid, lactic acid, gluconic acid; ketones; amino acids, such as glutamic acid (sodium monoglutaminate), but also more complex compounds such as antibiotics, such as penicillin, tetracyclin; enzymes; vitamins, such as riboflavin, B12, beta-carotene; hormones, which are difficult to produce synthetically.
Production of Sweeteners from Starch:
A “traditional” process for conversion of starch to fructose syrups normally consists of three consecutive enzymatic processes, viz. a liquefaction process followed by a saccharification process and an isomerization process. During the liquefaction process, starch is degraded to dextrins by an endo-amylase, preferably by an endo-amylase having a CBM, such as the polypeptide of the invention at pH values between 5.5 and 6.2 and at temperatures of 95-160° C. for a period of approx. 2 hours. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions).
After the liquefaction process the dextrins are converted into dextrose by addition of a glucoamylase (e.g., AMGTM) and a debranching enzyme, such as an isoamylase or a pullulanase (e.g., PromozymeTM). Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95° C.), and the liquefying endo-amylase activity is denatured. The temperature is lowered to 60° C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.
After the saccharification process the pH is increased to a value in the range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immmobilized glucoseisomerase (such as SweetzymeTM).
In an embodiment of a starch process of the invention, milled gelatinized whole grain raw material is broken down (hydrolyzed) into maltodextrins (dextrins) mostly of a DE higher than 4 using the polypeptide of the first aspect. The raw material is in one embodiment of the invention milled (whole) grain.
In an embodiment of the invention, enzymatic liquefaction is carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C., preferably 80-85° C., and the enzyme(s) is(are) added to initiate liquefaction (thinning), at least a polypeptide of the first aspect is added. Then the slurry is jet-cooked at a temperature between 95-140° C., preferably 105-125° C. to complete gelanitization of the slurry. Then the slurry is cooled to 60-95° C. and more enzyme(s), preferably comprising the polypeptide of the first aspect, is (are), added to finalize hydrolysis (secondary liquefaction). The liquefaction process is carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied whole grains are known as mash. The polypeptide of the first aspect may be added in effective amounts well known to the person skilled in the art.
In an aspect the process may comprise; a) contacting a starch substrate with a endo-amylase having a CBM, e.g., the polypeptide of the first aspect; b) incubating said starch substrate with said polypeptide and a fungal alpha-amylase and/or or a glucoamylase for a time and at a temperature sufficient to achieve liquefaction and saccharification of at least 90%, or at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% w/w of said starch substrate into fermentable sugars; c) fermenting to produce a fermentation product, d) optionally recovering the fermentation product.
In yet another aspect the process comprising liquefaction and/or hydrolysis of a slurry of gelatinized or granular starch, in particular liquefaction and/or hydrolysis of granular starch into a soluble starch hydrolysate at a temperature below the initial gelatinization temperature of said granular starch. In addition to being contacted with a polypeptide of the invention, e.g., the polypeptide of the first aspect, the starch may be contacted with an enzyme selected from the group consisting of; a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3). In an embodiment further a debranching enzyme, such as an isoamylase (E.C. 3.2.1.68) or a pullulanases (E.C. 3.2.1.41) may be added.
In an embodiment the process is conducted at a temperature below the initial gelatinization temperature. Preferably the temperature at which the processes are conducted is at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 43° C., at least 44° C., at least 45° C., at least 46° C., at least 47° C., at least 48° C., at least 49° C., at least 50° C., at least 51° C., at least 52° C., at least 53° C., at least 54° C., at least 55° C., at least 56° C., at least 57° C., at least 58° C., at least 59° C., or preferably at least 60° C. The pH at which the process is conducted may in be in the range of 3.0 to 7.0, preferably from 3.5 to 6.0, or more preferably from 4.0-5.0. In a preferred embodiment the process comprises fermentation, e.g., with a yeast to produce ethanol, e.g., at a temperature around 32° C., such as from 30 to 35° C. During the fermentation the ethanol content reaches at least 7%, at least 8%, at least 9%, at least 10% such as at least 11%, at least 12%, at least 13%, at least 14%, at least 15% such as at least 16% ethanol (w/w).
The starch slurry to be used in any of the above aspects may have 20-55% dry solids granular starch, preferably 25-40% dry solids granular starch, more preferably 30-35% dry solids granular starch. After being contacted with the endo-amylase having a CBM, e.g., the polypeptide of the first aspect at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids of the granular starch is converted into a soluble starch hydrolysate.
In another preferred embodiment the endo-amylase having a CBM, e.g, the polypeptide of the first aspect, is used in a process for liquefaction, saccharification of a gelatinized starch, e.g., but not limited to gelatinization by jet cooking. The process may comprise fermentation to produce a fermentation product, e.g., ethanol. Such a process for producing ethanol from starch-containing material by fermentation comprises: (i) liquefying said starch-containing material with a endo-amylase having a CBM, e.g., the polypeptide of the first aspect; (ii) saccharifying the liquefied mash obtained; (iii) fermenting the material obtained in step (ii) in the presence of a fermenting organism. Optionally the process further comprises recovery of the ethanol. The saccharification and fermentation may be carried out as a simultaneous saccharification and fermentation process (SSF process). During the fermentation the ethanol content reaches at least 7%, at least 8%, at least 9%, at least 10% such as at least 11%, at least 12%, at least 13%, at least 14%, at least 15% such as at least 16% ethanol.
The starch to be processed in the processes of the above aspects may in particular be obtained from tubers, roots, stems, legumes, cereals or whole grain. More specifically the granular starch may be obtained from corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, bean, banana or potatoes. Specially contemplated are both waxy and non-waxy types of corn and barley.
Compositions of the Invention
The invention also relates to a composition comprising the polypeptide of the first aspect. The composition may further comprise an enzyme selected from the group comprising of; a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (E.C. 3.2.1.2), a glucoamylase (E.C.3.2.1.3) and a pullulanases (E.C. 3.2.1.41). The glucoamylase may preferably be derived from a strain of Aspergillus sp., such as Aspergillus niger, or from a strain of Talaromyces sp. and in particular derived from Talaromyces leycettanus such as the glucoamylase disclosed in U.S. Pat. No. Re. 32,153, Talaromyces duponti and/or Talaromyces thermopiles such as the glucoamylases disclosed in U.S. Pat. No. 4,587,215 and more preferably derived from Talaromyces emersonii. Most preferably the glucoamylase is derived from Talaromyces emersonii strain CBS 793.97 and/or having the sequence disclosed as SEQ ID NO: 7 in WO 99/28448. Further preferred is a glucoamylase which has an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or even at least 95% homology to the aforementioned amino acid sequence. A commercial Talaromyces glucoamylase preparation is supplied by Novozymes A/S as Spirizyme Fuel.
Also preferred for a composition comprising the polypeptide of the first aspect and a glucoamylase are polypeptides having glucoamylase activity which are derived from a strain of the genus Trametes, preferably Trametes cingulata. Further preferred is polypeptides having glucoamylase activity and having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or even at least 95% homology with amino acids for mature polypeptide amino acids 1 to 575 of SEQ ID NO: 5 in U.S. Patent application No. 60/650,612.
Also preferred for a composition comprising the polypeptide of the first aspect and a glucoamylase are polypeptides having glucoamylase activity which are derived from a strain of the genus Pachykytospora, preferably Pachykytospora papyracea. Further preferred is polypeptides having glucoamylase activity and having at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or even at least 95% homology with amino acids for mature polypeptide amino acids 1 to 556 of SEQ ID NO: 2 in U.S. Patent application 60/650,612.
The composition described above may be used for liquefying and/or saccharifying a gelatinized or a granular starch, as well as a partly gelatinized starch, e.g., in a production of sweetener, or a fermentation process, such as for ethanol. A partly gelatinized starch is a starch which to some extent is gelatinized, i.e., wherein part of the starch has irreversibly swelled and gelatininized and part of the starch is still present in a granular state.
The composition described above may also comprise an acid fungal alpha-amylase present in an amount of 0.01 to 10 AFAU/g DS, preferably 0.1 to 5 AFAU/g DS, more preferably 0.5 to 3 AFAU/AGU, and most preferably 0.3 to 2 AFAU/g DS. The composition may be applied in any of the starch processes described above.
Production of Fermentation Products
From gelatinized starch: In this aspect the present invention relates to a process for producing a fermentation product, especially ethanol, from starch-containing material, which process includes a liquefaction step and separately or simultaneously performed saccharification and fermentation step(s). The fermentation product, such as especially ethanol, may optionally be recovered after fermentation, e.g., by distillation. Suitable starch-containing starting materials are listed in the section “Starch-containing materials” section below. Contemplated enzymes are listed in the “Enzymes”-section below. The fermentation is preferably carried out in the presence of yeast, preferably a strain of Saccharomyces. Suitable fermenting organisms are listed in the “Fermenting Organisms” section below.
A preferred process comprises a) contacting an aqueous starch slurry with a polypeptide comprising a first amino acid sequence having alpha-amylase activity and a second amino acid sequence comprising a carbohydrate-binding module, b) incubating said starch slurry with said polypeptide, c) fermenting to produce a fermentation product, and d) optionally recovering the fermentation product. Preferably the step b) is performed for a time and at a temperature sufficient to achieve conversion of at least 90% w/w of said starch substrate into fermentable sugars. Preferably the first amino acid sequence and/or second amino acid sequence of said polypeptide is derived from a bacterium. Said polypeptide may preferably be the hybrid of the first aspect.
The aqueous slurry may contain from 10-40 wt. %, preferably 25-35 wt. % starch-containing material. The slurry is heated to above the gelatinization temperature and bacterial and/or acid fungal alpha-amylase may be added to initiate liquefaction (thinning). The slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to an alpha-amylase in step (a) of the invention.
More specifically liquefaction may be carried out as a three-step hot slurry process. The slurry is heated to between 60-95° C., preferably 80-85° C., and alpha-amylase is added to initiate liquefaction (thinning). Then the slurry may be jet-cooked at a temperature between 95-140° C., preferably 105-125° C., for 1-15 minutes, preferably for 3-10 minute, especially around 5 minutes. The slurry is cooled to 60-95° C. and more alpha-amylase is added to finalize hydrolysis (secondary liquefaction). The liquefaction process is usually carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied whole grains are known as mash.
The saccharification in step may be carried out using conditions well know in the art. For instance, a full saccharification process may lasts up to from about 24 to about 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes at a temperature between 30-65° C., typically about 60° C., followed by complete saccharification during fermentation in a simultaneous saccharification and fermentation process (SSF). Saccharification is typically carried out at temperatures from 30-65° C., typically around 60° C., and at a pH between 4 and 5, normally at about pH 4.5.
The most widely used process in ethanol production is the simultaneous saccharification and fermentation (SSF) process, in which there is no holding stage for the saccharification, meaning that fermenting organism, such as yeast, and enzyme(s) may be added together. When doing SSF it is common to introduce a pre-saccharification step at a temperature above 50° C., just prior to the fermentation.
In accordance with the present invention the fermentation step (c) includes, without limitation, fermentation processes used to produce alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. Preferred fermentation processes include alcohol fermentation processes, as are well known in the art. Preferred fermentation processes are anaerobic fermentation processes, as are well known in the art.
From un-gelatinized starch: In this embodiment the invention relates to processes for producing a fermentation product from starch-containing material without gelatinization of the starch-containing material. In one embodiment a polypeptide of the invention, e.g., the hybrid enzyme of the first aspect, and optionally a glucoamylase is used during saccharification and fermentation. According to the invention the desired fermentation product, such as ethanol, can be produced without liquefying the aqueous slurry containing the starch-containing material. In one embodiment a process of the invention includes saccharifying milled starch-containing material below the initial gelatinization temperature in the presence of the hybrid enzyme of the first aspect and a glucoamylase to produce sugars that can be fermented into the desired fermentation product by a suitable fermenting organism.
A preferred process comprises a) contacting an aqueous granular starch slurry with a polypeptide comprising a first amino acid sequence having alpha-amylase activity and a second amino acid sequence comprising a carbohydrate-binding module, b) incubating said starch slurry with said polypeptide, c) fermenting to produce a fermentation product, and d) optionally recovering the fermentation product. Preferably the step b) is performed for a time and at a temperature sufficient to achieve conversion of at least 90% w/w of said starch substrate into fermentable sugars. Preferably the first amino acid sequence and/or second amino acid sequence of said polypeptide is derived from a bacterium. Said polypeptide may preferably be the hybrid of the first aspect.
The term “initial gelatinization temperature” means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50° C. and 75° C.; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this invention the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein and Lii, 1992, Starch/Stärke 44(12): 461-466.
Before step (a) a slurry of starch-containing material, such as granular starch, having 20-55 wt. % dry solids, preferably 25-40 wt. % dry solids, more preferably 30-35 wt. % dry solids of starch-containing material may be prepared. The slurry may include water and/or process waters, such as stillage (backset), scrubber water, evaporator condensate or distillate, side stripper water from distillation, or other fermentation product plant process water. Because the process of the invention is carried out below the gelatinization temperature and thus no significant viscosity increase takes place, high levels of stillage may be used if desired. In an embodiment the aqueous slurry contains from about 1 to about 70 vol. % stillage, preferably 15-60% vol. % stillage, especially from about 30 to 50 vol. % stillage.
The milled starch-containing material may be prepared by milling starch-containing material to a particle size of 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being subjected to a process of the invention at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dry solids of the starch-containing material is converted into a soluble starch hydrolysate.
The process of the invention is conducted at a temperature below the initial gelatinization temperature. Preferably the temperature at which step (a) is carried out is between 30-75° C., preferably between 45-60° C.
In a preferred embodiment step (a) and step (b) are carried out as a simultaneous saccharification and fermentation process. In such preferred embodiment the process is typically carried at a temperature between 28° C. and 36° C., such as between 29° C. and 35° C., such as between 30° C. and 34° C., such as around 32° C. According to the invention the temperature may be adjusted up or down during fermentation.
In an embodiment simultaneous saccharification and fermentation is carried out so that the sugar level, such as glucose level, is kept at a low level such as below about 3 wt. %, preferably below about 2 wt. %, more preferred below about 1 wt. %., even more preferred below about 0.5 wt. %, or even more preferred below about 0.1 wt. %. Such low levels of sugar can be accomplished by simply employing adjusted quantities of enzyme and fermenting organism. A skilled person in the art can easily determine which quantities of enzyme and fermenting organism to use. The employed quantities of enzyme and fermenting organism may also be selected to maintain low concentrations of maltose in the fermentation broth. For instance, the maltose level may be kept below about 0.5 wt. % or below about 0.2 wt. %.
The process of the invention may be carried out at a pH in the range between 3 and 7, preferably from 3.5 to 6, or more preferably from 4 to 5.
Starch-Containing Materials
Any suitable starch-containing starting material, including granular starch, may be used according to the present invention. The starting material is generally selected based on the desired fermentation product. Examples of starch-containing starting materials, suitable for use in a process of present invention, include tubers, roots, stems, whole grains, corns, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice peas, beans, or cereals, sugar-containing raw materials, such as molasses, fruit materials, sugar, cane or sugar beet, potatoes, and cellulose-containing materials, such as wood or plant residues. Contemplated are both waxy and non-waxy types of corn and barley.
The term “granular starch” means raw uncooked starch, i.e., starch in its natural form found in cereal, tubers or grains. Starch is formed within plant cells as tiny granules insoluble in water. When put in cold water, the starch granules may absorb a small amount of the liquid and swell. At temperatures up to 50° C. to 75° C. the swelling may be reversible. However, with higher temperatures an irreversible swelling called “gelatinization” begins. Granular starch to be processed may be a highly refined starch quality, preferably at least 90%, at least 95%, at least 97% or at least 99.5% pure or it may be a more crude starch containing material comprising milled whole grain including non-starch fractions such as germ residues and fibers. The raw material, such as whole grain, is milled in order to open up the structure and allowing for further processing. Two milling processes are preferred according to the invention: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein) and is often applied at locations where the starch hydrolysate is used in production of syrups. Both dry and wet milling is well known in the art of starch processing and is equally contemplated for the process of the invention.
The starch-containing material is milled in order to expose more surface area. In an embodiment the particle size is between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the milled starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen.
Fermentation Product
The term “fermentation product” means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferred fermentation processes used include alcohol fermentation processes, as are well known in the art. Preferred fermentation processes are anaerobic fermentation processes, as are well known in the art.
Fermenting Organisms
“Fermenting organism” refers to any organism, including bacterial and fungal organisms, suitable for use in a fermentation process and capable of producing desired a fermentation product. Especially suitable fermenting organisms are able to ferment, i.e., convert, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae. 
In a preferred embodiment the fermenting organism, e.g., the yeast, may be transformed with the polypeptide of the first aspect and applied in a process comprising; a) contacting a starch substrate with a fermenting organism cell transformed to express a polypeptide comprising a first amino acid sequence having alpha-amylase activity and a second amino acid sequence comprising a carbohydrate-binding module; b) holding said starch substrate with said yeast for a time and at a temperature sufficient to achieve conversion of at least 90% w/w of said starch substrate into fermentable sugars; c) fermenting to produce a fermentation product, e.g., ethanol, d) optionally recovering the fermentation product, e.g., ethanol. The steps a, b, and c are performed separately or simultaneously. In a preferred embodiment the first amino acid sequence and/or second amino acid sequence of said polypeptide is derived from a bacterium.
Materials and Methods
KNU amylolytic activity: The amylolytic activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue colour is formed, but during the break-down of the starch the blue colour gets weaker and gradually turns into a reddish-brown, which is compared to a coloured glass standard.
One Kilo Novo alfa Amylase Unit (KNU) is defined as the amount of enzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003 M Ca2+; and pH 5.6) dextrinizes 5.26 g starch dry substance Merck Amylum solubile. A folder AF 9/6 describing this analytical method in more detail is available upon request to Novozymes A/S, Denmark, which folder is hereby included by reference.
Endo activity assay: Endo endo-amylase activity may be determined using the Endo activity assay. 1 mL resuspended Phadebas substrate (0.25 tablets/mL 50 mM sodium acetate, 1 mM CaCl2, adjusted to pH 5.7) is incubated with 25 microL enzyme for 15 min at 40° C. with agitation. The reaction is stopped by addition of 0.5 mL 1 M NaOH and the mixture is centrifuged in a table centrifuge at 14,000 RPM. The absorbance of the supernatant at 620 nm is measured. The activity is determined by comparing to a standard with declared activity (BAN 480 L, 480 KNU/g).

Maltogenic amylase activity: One MANU (Maltogenic Amylase Novo Unit) may be defined as the amount of enzyme required to release one micromol of maltose per minute at a concentration of 10 mg of maltotriose (Sigma M 8378) substrate per ml of 0.1 M citrate buffer, pH 5.0 at 37° C. for 30 minutes (MANU unit further defined in U.S. Pat. No. 6,162,628, which is hereby incorporated by reference).

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations were performed using standard methods of molecular biology as described in Sambrook et al., 1989, Molecular cloning: A laboratory manual, Cold Spring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) “Current protocols in Molecular Biology”, John Wiley and Sons, 1995; Harwood, C. R. and Cutting, S. M. (eds.).
EXAMPLE 1
Construction of Hybrids Between an Endo-Amylase and the CBM from AMY1048
The amylase AMY1048 is a wild type Bacillus amylase made up of a catalytic fragment of 484 amino acid and in addition a CBM20 fragment of 101 aa. The DNA sequence coding the AMY1048 is included as SEQ ID NO:1 and the mature AMY1048 sequence is included as SEQ ID NO:2. In SEQ ID NO:1 the CBM is defined as amino acid residues 485 to 586 which correspond to nucleotides 1540-1845 in SEQ ID NO:2. The amylase including the CBM can be expressed from a construction similar to what have been described for other amylases, e.g., inserted into a vector under the control of a constitutive active promoter and flanked by the signal sequence (SEQ ID NO:15) and the terminator sequence of B. licheniformis endo-amylase.
Replacing the catalytic fragment of the AMY1048 endo-amylase with a catalytic domain of another endo-amylase, thus creating a hybrid of the CBM from AMY1048 and a new endo-amylase, is made by amplifying the DNA fragment coding the catalytic domain of the new amylase by PCR using two oligonucleotides. The sense oligonucleotide is in it's 5′end identical to the last 20 nucleotides of the DNA sequence coding for the signal sequence prior to the AMY1048 mature sequence and further in its 3′ end is identical to the first 20 nucleotides of DNA sequence coding the mature part of the desire amylase DNA. The antisense oligonucleotides are in its 5′ end identical to the antisense DNA of the first 20 nucleotides of the DNA sequence coding the CBM from AMY1048 and further in its 3′ end is identical to the antisense of the last 20 nucleotides of the DNA sequence coding the mature part of the desire amylase DNA.
Both the amplified amylase DNA and the vector hosting the AMY1048 amylase, is digested with Sac II and Sca I and the vector and PCR fragments ligated prior to transferring into Bacillus subtilis strain SHA273. In the primer sequences below the recognition sites of the restriction enzymes are indicated by underscore.
To construct a hybrid of the B. licheniformis endo-amylase (SEQ ID NO:35) and the CBM20 from B. flavothermus amylase the following oligonucleotides were used by the present inventors:







Sense:


(P1s SEQ ID NO: 19)


5′-ctcattctgcagccgcggcagcaaatcttaatgggacgct-3′.


 


Antisense:


(P1as SEQ ID NO: 20)


5′- atttgggaagtagtacttattctttgaacataaattgaaa-3′.






The resulting DNA sequence coding the mature polypeptide and the amino acid sequence of the mature polypeptide are included as SEQ ID NO:3 and SEQ ID NO:4 respectively
To construct a hybrid of the LE429 variant of B. licheniformis endo-amylase (SEQ ID NO:41) and the CBM20 from B. flavothermus amylase the following oligonucleotides were used:









Sense:



(P2s SEQ ID NO: 21)



5′-ctcattctgcagccgcggcagtaaatggcacgctgatgca-3′



 



Antisense:



(P2as SEQ ID NO: 22)



5′-atttgggaagtagtacttatttttggaacataaattgaaa-3′






The resulting DNA sequence coding the mature polypeptide and the amino acid sequence of the mature polypeptide are included as SEQ ID NO:5 and SEQ ID NO:6 respectively
To construct a hybrid of the B. stearothermophilus endo-amylase (SEQ ID NO:36) and the CBM20 from B. flavothermus amylase the following oligonucleotides were used:









Sense:



(P3s SEQ ID NO: 23)



5′-ctcattctgcagccgcggcagcaccgtttaacggctttaa-3′



 



Antisense:



(P3as SEQ ID NO: 24)



5′-atttgggaagtagtacttattttaggaacccaaaccgaaa-3′






The resulting DNA sequence coding the mature polypeptide and the amino acid sequence of the mature polypeptide are included as SEQ ID NO:7 and SEQ ID NO:8 respectively
To construct a hybrid of a variant of the alkaline Bacillus sp. SP722 endo-amylase (SEQ ID NO:38) and the CBM20 from B. flavothermus amylase the following oligonucleotides were used:







Sense:


(P4s SEQ ID NO: 25)


5′ctcattctgcagccgcggcacatcataatgggacaaatgg-3′.


 


Antisense:


(P4as SEQ ID NO: 26)


5′- atttgggaagtagtacttatccatttgtcccattatgatg-3′.






The resulting DNA sequence coding the mature polypeptide and the amino acid sequence of the mature polypeptide are included as SEQ ID NO:9 and SEQ ID NO:10 respectively.
To construct a hybrid of a variant of the alkaline Bacillus species AA560 endo-amylase (SEQ ID NO:40) and the CBM20 from B. flavothermus amylase the following oligonucleotides were used:







Sense:


(P5s SEQ ID NO: 27)


5′-ctcattctgcagccgcggcacaccataatggtacgaacgg-3′


 


Antisense:


(P5as SEQ ID NO: 28)


5′- atttgggaagtagtacttattttgtttacccaaatagaaa-3′






The resulting DNA sequence coding the mature polypeptide and the amino acid sequence of the mature polypeptide are included as SEQ ID NO:11 and SEQ ID NO:12 respectively.
To construct a hybrid of a variant of the Bacillus amyloliquefaciens endo-amylase (SEQ ID NO:37) and the CBM20 from B. flavothermus amylase the following oligonucleotides were used:







Sense:


(P6s SEQ ID NO: 29)


5′-ctcattctgcagccgcggcagtaaatggcacgctgatgca-3′


 


Antisense:


(P6as SEQ ID NO: 30)


5′- atttgggaagtagtacttatttttggaacataaatggaga-3′






The resulting DNA sequence coding the mature polypeptide and the amino acid sequence of the mature polypeptide are included as SEQ ID NO:13 and SEQ ID NO:14 respectively.
The above described hybrid enzymes was expressed by B. subtilis growing in shake flasks for 72 hours at and secreted into the supernatant. The presence of hybrid enzyme in the supernatant was demonstrated by SDS-PAGE.
EXAMPLE 2
Construction of a Hybrid Amylase with Carbohydrate Binding Domain
The catalytic fragment of the B. flavothermus endo-amylase, AMY1048 can further be divided into the central AB-domain harboring the catalytic center and a C domain c-terminal to the catalytic domain but prior to the CBM. In SEQ ID NO:2 the catalytic core domain consist of the first 397 amino acid residues, the C domain is defined as the amino acid residues from 398 to 484 and the CBM is defined as amino acid residues 485 to 586. In SEQ ID NO:1 the signal sequence is encoded by nucleotide 1 to 87, the catalytic core domain is encoded by nucleotide 88-1278, the C domain is encoded by the nucleotides 1279-1539, and the CBM is encoded by nucleotides 1540-1845.
The amylase including the CBM can be expressed from a vector construction similar to what have been described in WO 00/60060 in example 4—i.e., the amylase gene is inserted into a vector under the control of a amylase promoter and flanked by the signal sequence and the terminator sequence of B. licheniformis endo-amylase.
As an alternative to harboring the gene on a plasmid, the cassette including the DNA sequence coding for the antibiotic marker, promoter, signal sequence, the mature protein and the terminator can be integrated into the genome of the B. subtilis by homologous in-vivo crossover, by flanked upstream and downstream genomic DNA with high similarity to a non-essential part of the B. subtilis DNA. Useful DNA regions could be the pectate lyase or the endo-amylase loci. In this example the AMY1048 and the hybrid is inserted into the amylase loci in opposite direction relative to the original B. subtilis amylase.
The catalytic core domain of the AMY1048 endo-amylase was replaced with a catalytic core domain of the Bacillus stearothermophilus (BSG) endo-amylase, thus creating a hybrid of the C-domain and the CBM from AMY1048 and the catalytic core domain from the new endo-amylase.
The DNA fragment coding the catalytic core of the B. stearothermophilus amylase (SEQ ID NO:36) was amplified by PCR using two oligonucleotides. The sense oligonucleotides were in its 5′ end identical to the last 20 nucleotides of the DNA sequence (SEQ ID NO:15) coding for the signal sequence prior the AMY1048 mature sequence (SEQ ID NO:1) and further in its 3′ end identical to the first 20 nucleotides of DNA sequence coding the mature part of the desire amylase DNA. The antisense oligonucleotides were in its 5′ end identical to the antisense DNA of the first 20 nucleotides of the DNA sequence coding the C-domain from AMY1048 and further in its 3′ end was identical to the antisense of the last 20 nucleotides of the DNA sequence coding the catalytic core of the BSG amylase DNA.
To construct a hybrid of the B. stearothermophilus endo-amylase core domain and C-domain and the CBM20 from B. flavothermus amylase the following oligonucleotides were used by the present inventors:







Sense:


(P7s SEQ ID NO: 31)


5′-ctcattctgcagccgcggcagcaccgtttaacggctttaa-3′.


 


Antisence:


(P7as SEQ ID NO: 32)


5′-atatagtcgtgctgtgttccgtaagcataatccctgcgcg-3′.






To facilitate genome integration, a 5 kB fragment upstream from of the signal sequence and into the amylase genome sequence is made by PCR using the AMY1048 genomic construction as template, and the inverse primer of the antisense primer and the genome specific primer : 5′-ctgcatcagggctgcggcatcc-3; (P8 SEQ ID NO:33).
Another fragment from the termination of the gene and upstream of the genomic B. subtilis amylase is made by PCR using the AMY1048 genomic construction as template, and the inverse primer of the sense primer and the genome specific primer: 5′-ctgcatcagggctgcggcatcc-3′; (P9 SEQ ID NO:34).
Taking advantages of the 40 by overlap, the three PCR fragments were assembled by PCR and the resulting product amplified in another PCR using the genome specific primers, prior to transferring into Bacillus subtilis strain SHA273 (described in WO 92/11357 and WO 95/10603).
The resulting DNA sequence coding the mature polypeptide and the mature polypeptide are included as SEQ ID NO:17 and SEQ ID NO:18 respectively.
The hybrid enzyme was expressed by B. subtilis growing in PS1 media in shake flasks for 72 hours at 37° C. and secreted into the supernatant. The presence of hybrid enzyme in the supernatant was demonstrated by SDS-PAGE.
EXAMPLE 3
Determination of Exo-Endo Improvement Factor (EIF)
EIF is the measure of an increment of the exo/endo ratio relative to a parent enzyme, i.e., EIF=(exo/endo of variant)/(exo/endo of parent enzyme). An enzyme has an increase in exo/endo ratio compared to its parent enzyme if EIF>1. EIF may be based on one of the following methods.
EIF1 Endo activity assay: The Phadebas Amylase Test (Pharmacia Diagnostics) is run according to the supplier's recommendations and the endo units calculated from the supplied formula where the natural logarithm to the activity equals N, where N=A+square root [B+C*ln(Abs)]. Abs is the absorbance at 620 nm, A=−13.3235, B=243.3293, and C=26.73797
Exo activity assay: 50 microL of 50 mM sodium citrate, 5 mM CaCl2, pH 6.5 is mixed with 25 microL of enzyme in the same buffer and 25 microL Betamyl substrate (Betamyl Method, Megazyme) dissolved according to suppliers recommendations. The assay mix is incubated for 30 min. at 40° C. and the reaction stopped by adding 150 microL 4% (w/w) Trizma base (Tris(hydroxymethyl)-aminomethane). The activity is expressed directly as the absorbance at 420 nm measured using a microtiter plate reader.
EIF2 Endo activity assay: 1 mL resuspended Phadebas substrate (Pharmacia Diagnostics) (0.25 tablets/mL 50 mM so-dium acetate, 1 mM CaCl2, adjusted to pH 5.7) is incubated with 25 microL enzyme for 15 min at 40° C. with agitation. The reaction is stopped by addition of 0.25 mL 1 M NaOH and the mix-ture is centrifuged in a table centrifuge at 14,000 RPM. The absorbance of the supernatant at 620 nm is measured. The activity is determined by comparing to a standard with declared activity (BAN 480 L, 480 KNU/g).
Exo activity assay: 900 microL 3.3% solubilized waxy maize starch (3.3% starch is boiled in 50 mM sodium acetate, 1 mM CaCl2, pH 5.7 for 5 min and cooled to 40° C.) is incubated with 100 microL enzyme at 40° C. with stirring. After appropriate reaction time the remaining starch is precipitated by addition of 450 microL 4° C. 96% ethanol. The precipitate is immediately removed by centrifugation at 3000 G for 20 min. The total carbohydrate in the supernatant is determined by mixing 200 microL supernatant with 50 microL 2% tryptophan and 900 microL 64% sulphuric acid. The mixture is heated for 15 min at 95° C. and the absorbance at 630 nm is measured after cooling to room tem-perature. The activity is determined by comparing with the absorbance of glucose standards in the same assay. One unit is defined as the amount of enzyme that at initial rates liberates 1 mg oligomeric products (products that are not precipitated by ethanol) per min.
EXAMPLE 4
Liquefaction and Saccharification with an Endo-Amylase with a CBM
This example illustrates the conversion of granular wheat starch into glucose using a bacterial endo-amylase with a CBM (SEQ ID NO:4) or the same bacterial endo-amylase without CBM (SEQ ID NO:35) together with a glucoamylase and an acid fungal amylase. A slurry with 33% dry solids (DS) granular starch was prepared by adding 247.5 g of wheat starch under stirring to 502.5 ml of water. The pH was adjusted with HCl to 4.5. The granular starch slurry was distributed to 100 ml Erlenmeyer flasks with 75 g in each flask. The flasks were incubated with magnetic stirring in a 60° C. water bath. At zero hours the enzyme activities given in table 1 were dosed to the flasks. Samples were withdrawn after 24, 48 and 73 and 94 hours. The enzyme levels used were endo-amylase +/−CBM 100 KNU/kg DS, glucoamylase 200 AGU/kg DS, acid fungal alpha-amylase 50 AFAU/g DS.
Total dry solids starch was determined using the following method. The starch was completely hydrolyzed by adding an excess amount of endo-amylase (300 KNU/Kg dry solids) and placing the sample in an oil bath at 95° C. for 45 minutes. Subsequently the samples were cooled to 60° C. and an excess amount of glucoamylase (600 AGU/kg DS) was added followed by incubation for 2 hours at 60° C.
Soluble dry solids in the starch hydrolysate were determined by refractive index measurement on samples after filtering through a 0.22 microM filter. The sugar profile was determined by HPLC. The amount of glucose was calculated as DX. The results are shown in tables 2 and 3.







TABLE 2







Soluble dry solids as percentage of total dry substance at


100 KNU/kg DS endo-amylase dosage.











Enzyme
24 hours
48 hours
73 hours
94 hours














Endo-amylase
83.7
87
89.7
90.3


Endo-amylase + CBM
87.2
89.7
91.5
92.3
















TABLE 3







The DX of the soluble hydrolysate at


100 KNU/kg DS endo-amylase dosage.











Enzyme
24 hours
48 hours
73 hours
94 hours





Endo-amylase
72.0
82.0
83.8
83.8


Endo-amylase + CBM
76.7
87.0
87.5
87.5









EXAMPLE 5
Effective Dosage
The “effective dosage” of the amylase in question is defined as the dosage resulting in a reduction in firmness of more than 10%, e.g., of between 10 and 20%, compared to the firmness of a bread without enzymes (the control). The reduction in firmness is measured after storage for 14 days in inert atmosphere at room temperature.
Tolerance towards overdosing is measured by using the Elasticity Loss Ratio=ELR. ELR is measured day 1 after baking or later, such as day 5, day 10 or as in the example below after 14 days storage and is defined then as follows:

ELR %=(Elasticitycontrol day 14−Elasticityamylase day 14×100)/Elasticitycontrol day 14 

In combination with 450 MANU/kg flour Novamyl® the tolerance towards overdosing is measured:

ELRN%=(ElasticityNovamyl day 14−ElasticityNovamyl+amylase day 14×100)/ElasticityNovoamyl day 14 

If the amylase is overdosed the ELR and/or ELRN will be >5%.
Baking Process
Bread is baked according to the sponge & dough method.










Sponge, ingredients as % on flour basis


















Soya oil
2.5



SSL
0.38



Yeast
5



Wheat flour
60



Water
62




















Dough, ingredients as % on flour basis


















Ascorbic acid
to be optimized for each flour



ADA
20 ppm



Salt
 2



Sirup
7 (dry substance)



Water
to be optimized for each flour



Wheat flour
40



Calcium propionate + enzymes
 0.25










The sponge ingredients yeast, water, flour, SSL and oil are mixed at 90 rpm for 1 minute, 150 rpm for 4 minutes. The sponge is set for fermentation for 3 hours at 27° C. and 86% RH.
The sponge is added the dough ingredients and mixed to a dough at 90 rpm for 1 minute and at 150 rpm for 14 minutes. The dough is scaled into pieces of 340 g each and rested for 10 minutes.
The dough portions are sheeted and molded followed by fermentation at 55 minutes at 42° C. and 86% RH. The doughs are baked at 225° C. for 15 minutes. The baked bread are cooled and stored until analysis.
Bread is baked with the CBM-hybrid enzyme and with the corresponding enzyme without a CBM. The effective dose is determined with and without addition of Novamyl® at 450 MANU/kg flour. Firmness and elasticity of a bread are measured by the TA.XT2 texture analyzer according to AACC method 74-09.
The effective dosage of the CBM-hybrid enzyme is determined and a new set of bread is baked with 3 and 5 times the effective dosage with and without addition of Novamyl® at 450 MAN U/kg flour.
The ELR is measured after 14 days of storage, and it is found that the ELR as well as the ELRN is less than 5% for the amylase with CBM dosed 5 times the effective dosage whereas it is more than 5% for the corresponding enzymes without addition of the CBM dosed 3 times the effective dose.
EXAMPLE 6
Determination of ELR for Selected Variants
Example 6 was performed as described in Example 5 except that a dosage of 500 MANU/kg flour was used.
Two variants of a hybrid comprising the alkaline Bacillus species AA560 endo-amylase (SEQ ID NO:40) and the CBM20 from the B. flavothermus amylase (residues 485 to 586 in SEQ ID NO:2) were used: The variant BE1 comprising the following alterations in the amylase sequence: R118K, D183*, G184*, N195F, R320K, R458K, N33S, D36N, K37L, E391I, Q394R, K395D, T452Y and N484P, and the variant BE2 comprising of the following alterations in the amylase sequence: R118K, D183*, G184*, N195F, R320K, R458K and N484P.







TABLE 1







Application of hybrid-amylase (1 mg/kg flour) without Novamyl













Firmness





Firmness on
reduction in %
Elasticity


Treatment
day15 (g)
of control day15
g/g
ELR %





Control
794

39.9



BE1
382
51
47.0
−17.0


BE2
313
61
46.6
−16.8
















TABLE 2







Application of hybrid-amylase in combination with Novamyl













Firmness





Firmness
reduction



on
in % of
Elasticity


Treatment
Day15 (g)
control day15
g/g
ELR %





Control
706

40.8



BE1 0.5 mg/kg flour
316
55
46.9
−4.5


BE1 1 mg/kg flour
239
66
47.0
−4.9


BE2 0.5 mg/kg flour
315
55
47.0
−4.9


BE2 1 mg/kg flour
225
68
47.5
−6.0


Only Novamyl ® 500
452

44.8


MANU/kg flour









EXAMPLE 7
Batter Cake
Batter cake dough was prepared with hybrids BE1, BE2, the Bacillus amylase shown in SEQ ID NO:40 (CD donor homologue) and the Bacillus amylases SEQ ID NO:2 (CBM donor).
The dough was made from a commercial batter cake mix “Tegral Allegro” from Puratos consisting of wheat flour, sugar, baking powder, emulsifier (mono- and diglycerides of fatty acids). The cake mix, enzyme (4 mg/kg flour) and water was place in a bowl and beat with a spatula, Bear AR 5 A-Vari-mixer, at third speed until a smooth homogeneous mixture was obtained (approximately 2 minutes). Molds were filled with 300 g dough and baked at 180° C. for 45 minutes. The baked cakes were cooled at room temperature for 30 minutes and packed in nitrogen before storage at room temperature until analysis.
Mobility of free water was determined using low field NMR as described by Chen, Long, Ruan and Labuza, 1997, Nuclear Magnetic Resonance Studies of water Mobility in bread during Storage. Lebensmittel Wissenschaft and Technologie 30: 178-183.
Hardness and cohesiveness was measured according to the method described in Food Texture and viscosity, 2nd edition, Malcolm Bourne, Food Science and Technology, International Series, Academic Press, page 182-186.
All data were measured after 14 days. The following results were obtained:













Treatment
Hardness units
Cohesiveness units
Mobility units







Reference
1485
34
4148


BE1 9.5 KNU/kg
1482
35
4655


flour


Amyl1
1702
35
4811


9.5 KNU/kg flour


BE3 9.5 KNU/kg
1217
34
4797


flour


BAN (SEQ ID
1456
32
4423


NO: 37)


9.5 KNU/kg flour









Based on the above data the following parameters (I)-(III) were calculated:

Cohesiveness reduction %=(CohesivenessReference−Cohesivenessamylase)×100%/CohesivenessReference   (I):

dHardness=HardnessReference−HardnessAmylase   (II):

dMobility=MobilityAmylase−MobilityReference   (III):















Cohesiveness




Treatment
Reduction %
dHardness units
dMobility units


















Reference





BE1 9.5 KNU/kg
−3
3
507


flour


Amyl1
−3
−217
663


9.5 KNU/kg flour


BE3
0
268
649


9.5 KNU/kg flour


BAN (SEQ ID
5.8
20
275


NO: 37)


9.5 KNU/kg flour









Amyl1 is identical to the amylase of SEQ ID NO: 40 with the following substitutions: R118K, D183*, G184*, N195F, R320K, R458K, N33S, D36N, K37L, E391I, Q394R, K395D, T452Y and N484P, using the numbering of SEQ ID NO: 40.
EXAMPLE 8
Sponge and Dough
Bread was baked according to the sponge & dough method. Bread was stored at room temperature for 14 days until analysis. Hardness and cohesiveness was measured according to the method described in Food Texture and viscosity, 2 edition, Malcolm Bourne, Food Science and Technology, International Series, Academic Press, page 182-186, and mobility of free water was determined using low field NMR as described by Chen, Long, Ruan and Labuza, 1997, Nuclear Magnetic Resonance Studies of water Mobility in bread during Storage. Lebensmittel Wissenschaft and Technologie 30: 178-183. Three amylases were used; the variants BE1 and BE3 and the Bacillus amylase SEQ ID NO:2 (CBM donor). The variant BE3 has a the catalytic domain having the amino acid sequence as shown in SEQ ID NO: 37 and comprise one or more, e.g., such as all of the following alterations: S31A, D32N, I33L, E178*, G179*, N190F, K389I, K392R, E393D, V508A and a CBM having the amino acid sequence shown as amino acid residues 485 to 586 in SEQ ID NO:2.
All data were measured after 14 days. The following results were obtained:














Hardness
Cohesiveness
Mobility


Treatment
units
units
units







Reference
400
38
6435


Novamyl 300
272
48
6234


MANU/kg flour


BE3 0.05 mg/kg flour +
256
48
7365


Novamyl 300


MANU/kg flour


BAN (SEQ ID
207
45
7354


NO: 37)


0.05 mg/kg flour +


Novamyl 300


MANU/kg flour


BE3 0.15 mg/kg flour
223
48
6886


BE1 0.5 mg/kg flour
311
41
7152









Based on the above data the following parameters (I)-(VI) were calculated:
For treatments without Novamyl®

Cohesiveness reduction %=(CohesivenessReference−Cohesivenessamylase)×100%/CohesivenessReference   (I):

dHardness=HardnessReference−HardnessAmylase   (II):

dMobility=MobilityAmylase−MobilityReference   (III):

For treatments with Novamyl®

Cohesiveness reduction %=(CohesivenessNovamyl−Cohesivenessamylase+Novamyl)×100%/CohesivenessNovamyl   (IV):

dHardness=HardnessNovamyl−HardnessAmoase+Novamyl   (V):

dMobility=MobilityAmylase+Novoamyl−MobilityNovoamyl   (VI):















Cohesiveness
dHardness
dMobility


Treatment
reduction %
units
units


















Reference





Novamyl 300


MANU/kg flour


BE3 0.05 mg/kg flour +
0
16
1131


Novamyl 300


MANU/kg flour


BAN (SEQ ID
6.3
65
1120


NO: 37)


0.05 mg/kg flour +


Novamyl 300


MANU/kg flour


BE3 0.15 mg/kg flour
−26
177
451


BE1 0.5 mg/kg flour
−7.9
89
717









EXAMPLE 9
Determination of Thermostablity
The thermostability was determined at 60, 65 or 70° C. for 30 minutes in a 50 mM NaOAc, 1 mM CaCl2 buffer at pH 5.7. The samples was cooled down and the residual activity was measured using the Phadebas method as describe in section Materials and Methods except that the determination took place at 50° C. The residual activity (R.A.) can be calculated according to the following equation: R.A. (%)=[Abs (heat treated)−Abs (blank)]/[Abs (heat treated at 60° C.)−Abs (blank)]*100%.
The following results were obtained:










Residual activity for Fungamyl, a well-known fungal baking


amylase from A. oryzae, and to hybrid enzymes of the invention.












Enzyme
60° C.
65° C.
70° C.
















Fungamyl
100
4
2



BE1
100
78
67



BE3
100
80
27

## Claims

1. A hybrid polypeptide comprising:
a) a first amino acid sequence having endo-amylase activity, and
b) a second amino acid sequence comprising a carbohydrate-binding module, wherein the hybrid polypeptide has at least 90% identity to any amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.

2. The hybrid polypeptide of claim 1, which has at least 90% identity to the amino acid sequence of SEQ ID NO: 4.

3. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 4 in no more than 10 positions.

4. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 4 in no more than 5 positions.

5. The hybrid polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO: 4.

6. The hybrid polypeptide of claim 1, which has at least 90% identity to the amino acid sequence of SEQ ID NO: 6.

7. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 6 in no more than 10 positions.

8. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 6 in no more than 5 positions.

9. The hybrid polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO: 6.

10. The hybrid polypeptide of claim 1, which has at least 90% identity to the amino acid sequence of SEQ ID NO: 10.

11. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 10 in no more than 10 positions.

12. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 10 in no more than 5 positions.

13. The hybrid polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO: 10.

14. The hybrid polypeptide of claim 1, which has at least 90% identity to the amino acid sequence of SEQ ID NO: 12.

15. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 12 in no more than 10 positions.

16. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 12 in no more than 5 positions.

17. The hybrid polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO: 12.

18. The hybrid polypeptide of claim 1, the hybrid polypeptide has at least 90% identity to the amino acid sequence of SEQ ID NO: 14.

19. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 14 in no more than 10 positions.

20. The hybrid polypeptide of claim 1, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 14 in no more than 5 positions.

21. The hybrid polypeptide of claim 1, which comprises the amino acid sequence of SEQ ID NO: 14.

22. A composition which comprises flour and a hybrid polypeptide of claim 1.

23. A dough- or bread-improving additive in the form of a granulate or agglomerated powder comprising a hybrid polypeptide of claim 1.

24. A process for producing a baked product, comprising baking a dough in the presence of a hybrid polypeptide of claim 1.

25. The process of claim 24, wherein the baked product is a bread.

26. A process for saccharifying starch, comprising treating a starch with a hybrid polypeptide of claim 1.

27. A process of producing a fermentation product, comprising
(a) liquefying a starch-containing material with a hybrid polypeptide of claim 1 to produce a liquefied mash;
(b) saccharifying the liquefied mash to produce a saccharified material;
(c) fermenting the saccharified material in the presence of a fermenting organism.

28. The process of claim 27, further comprising recovery the fermentation product.

29. The process of claim 27, wherein the fermentation product is ethanol.

30. A hybrid polypeptide comprising:
a) a first amino acid sequence having endo-amylase activity, and
b) a second amino acid sequence comprising a carbohydrate-binding module, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 8 in no more than 10 positions.

31. The hybrid polypeptide of claim 30, wherein the amino acid sequence of the hybrid polypeptide differs from the amino acid sequence of SEQ ID NO: 8 in no more than 5 positions.

32. The hybrid polypeptide of claim 30, which comprises the amino acid sequence of SEQ ID NO: 8.

33. A composition which comprises flour and a hybrid polypeptide of claim 30.

34. A dough- or bread-improving additive in the form of a granulate or agglomerated powder comprising a hybrid polypeptide of claim 30.

35. A process for producing a baked product, comprising baking a dough in the presence of a hybrid polypeptide of claim 30.

36. The process of claim 35, wherein the baked product is a bread.

37. A process for saccharifying starch, comprising treating a starch with a hybrid polypeptide of claim 30.

38. A process of producing a fermentation product, comprising
(a) liquefying a starch-containing material with a hybrid polypeptide of claim 30 to produce a liquefied mash;
(b) saccharifying the liquefied mash to produce a saccharified material;
(c) fermenting the saccharified material in the presence of a fermenting organism.

39. The process of claim 38, further comprising recovery the fermentation product.

40. The process of claim 38, wherein the fermentation product is ethanol.